Dynamics and stability in maturation of a T=4 virus

Abstract

Nudaurelia capensis omega virus is a T=4, icosahedral virus with a bipartite, positive-sense RNA genome. Expression of the coat protein gene in a baculovirus system was previously shown to result in the formation of procapsids when purified at pH 7.6. Procapsids are round, porous particles (480 A diameter) and have T=4 quasi-symmetry. Reduction of pH from 7.6 to 5.0 resulted in virus-like particles (VLP(5.0)) that are morphologically identical with authentic virions, with an icosahedral-shaped capsid and a maximum dimension of 410 A. VLP(5.0) undergoes a maturation cleavage between residues N570 and F571, creating the covalently independent gamma peptide (residues 571-641) that remains associated with the particle. This cleavage also occurs in authentic virions, and in each case, it renders the morphological change irreversible (i.e., capsids do not expand when the pH is raised back to 7.6). However, a non-cleavable mutant, N570T, undergoes the transition reversibly (NT(7.6)<-->NT(5.0)). We used electron cryo-microscopy and three-dimensional image reconstruction to study the icosahedral structures of NT(7.6), NT(5.0), and VLP(5.0) at about 8, 6, and 6 A resolution, respectively. We employed the 2. 8-A X-ray model of the mature virus, determined at pH 7.0 (XR(7.0)), to establish (1) how and why procapsid and capsid structures differ, (2) why lowering pH drives the transition, and (3) why the non-cleaving NT(5.0) is reversible. We show that procapsid assembly minimizes the differences in quaternary interactions in the particle. The two classes of 2-fold contacts in the T=4 surface lattice are virtually identical, both mediated by similarly positioned but dynamic gamma peptides. Furthermore, quasi and icosahedral 3-fold interactions are indistinguishable. Maturation results from neutralizing the repulsive negative charge at subunit interfaces with significant differentiation of quaternary interactions (one 2-fold becomes flat, mediated by a gamma peptide, while the other is bent with the gamma peptide disordered) and dramatic stabilization of the particle. The gamma peptide at the flat contact remains dynamic when cleavage cannot occur (NT(5.0)) but becomes totally immobilized by noncovalent interactions after cleavage (VLP(5.0)).

Figure 1

(a) Schematic diagram of the subunit organization in the T=4 NωV capsid. The four subunits in each asymmetric unit are A (blue), B (red), C (green), and D (yellow). White symbols identify icosahedral symmetry axes and black symbols identify quasi two-fold (ellipses) and quasi three-fold (triangle) axes. Dimer interfaces at the quasi two-folds occur with bent (A-B) or flat (C-D) conformations. Well-ordered C-terminal helices in the C and D subunitsstabilize the flat C-D interface. (b) Ribbon diagram of the C subunit tertiary structure showing the three-domain organization present in all subunits. Maturation cleavage occurs in the innerhelical domain between residues Asn570 and Phe571. The γ-peptide (colored blue to red from its N- to C-termini) includes a 3-helix, 2-loop motif. The molecular switch (switch helix) only occurs in the C and D subunits. (c) Procapsid-capsid transitions in wild type and mutant particles. In WT virus, a drop in pH rapidly converts procapsids to uncleaved capsids, followed by slower maturation cleavage, which locks the particle in the capsid conformation. In the cleavage-defective mutant, N570T (NT), particle transitions are reversible.

Figure 2

8-Å cryoEM reconstruction of the NT procapsid at pH 7.6. (a) Global view of the procapsid outer surface (color coded as in Fig. 1a). (b) Schematic representations of the quasi 2-fold A-B and C-D dimers. (c) Stereo view of the reconstructed density (grey isosurface) for the A-B dimer with an atomic model of each subunit derived from the capsid crystal structure fitted independently into the corresponding density. (d) Same as (c) for the C-D dimer. Similarity of the procapsid A-B and C-D dimer interfaces is apparent because the Ig-like domains maintain a 13 Å separation in both dimers.

Figure 3

Pseudo-atomic model of the inner surface of NT_7.6 centered on the quasi-3-fold trefoil. (a) Stereo, ribbon diagram, of the modeled, inner helical domains. Filled ellipses identify A-B dimer quasi two-folds (at left and right) and the C-D dimer quasi two-fold (at bottom). The trefoil of helices was generated with helix interactions of the A and B subunits observed in XR_7.0. While those same interactions do not occur in A-C and C-B in XR_7.0, this 3-fold enforced model fits the trefoil density exceptionally well as shown in (b). The switch helices at the AB and CD interfaces are also shown in the model (purple), although density for these is either completely absent or very weak owing to their dynamic character. (b) Same as (a) with the pseudo-atomic model fit into the NT_7.6 density map.

Figure 4

6-Å cryoEM reconstruction of VLP_5.0. (a) Global view of the VLP_5.0 structure (color coded as in Fig. 1a). (b) Schematic representations of the quasi 2-fold A-B and C-D dimers. (c) Stereo view of the reconstructed density (grey isosurface) for the A-B dimer with an atomic model of each subunit derived from the capsid crystal structure fitted independently into the corresponding density. (d) Same as (c) for the C-D dimer. Unlike the procapsid where the Ig-like domains are 13 Å apart in both dimers (Fig. 2), in capsids, the Ig-like domains in the C-D dimer are only 5 Å apart.

Figure 5

Comparison of 6-Å electron density for uncleaved (NT_5.0) and cleaved (VLP_5.0) capsids at pH 5.0. The first column shows the cryoEM map, the second and third columns are stereo view of the same map superimposed with the XR_7.0 model. (a) and (b) Comparison of density for NT_5.0 and VLP_5.0 around the 3-fold axis. The green arrows point out there are more density in the middle of the trimer in NT_5.0 compared with the VLP_5.0 structure. Red arrows highlight the switch helices densities which is missing at the flat contact in NT_5.0 and strong VLP_5.0. This is consistent with reduced dynamic character of the switch helices after maturation cleavage. (c) and (d) Comparison of 10-helix bundle at the 5-fold axes in NT_5.0 and VLP_5.0. There is no density for residues 44-55 from the B subunit in NT_5.0 and density for the last 8 residues of the A helix (592-599) is missing. In contrast, in VLP_5.0 the XR_7.0 model fits the density extremely well, which illustrates that cleavage dramatically reduces the mobility of these helices.

Figure 6

A diagram comparing portions of the g peptide that are visible in the electron density for VLP_5.0 and NT_5.0. A, B, C and D represent the four independent subunits in the icosahedral asymmetric unit of the virus. Ordered and disordered regions were represented by the solid and dotted lines respectively. The cleaved VLP_5.0 contains more ordered regions than the NT_5.0 structure.